The present invention relates to a method for generating DNA nested deletions by in vitro reactions.
The present invention also relates to a kit for use in said method for generating DNA nested deletions.
In genetic engineering technology, a desired DNA fragment is often manipulated by being insertedinto a vector. In such a case, it may be necessary to prepare the DNA insert in a length suitable for maniuplation.
For example, it is important to insert a DNA fragment of a suitable length into a vector, particularly in DNA sequencing techniques which can determine only a limited length of sequence at once. More specifically, the current DNA sequencing techniques can determine a sequence of at most about 1000 bases, usually only 300 to 400 bases at once. Therefore, longer fragments are sequenced by 1) subcloning, 2) primer walking, and 3) nested deletion or other methods. Among them, the nested deletion method is a technique which generates a number of nested deletions from a fixed site and it is more promising than the former two methods because a variety of deletion products can be conveniently obtained, as well as for other reasons. The nested deletion method hitherto known is performed in vitro by using exonuclease such as ExoIII or in vivo by using the terminal repeat of a transposon and transposase.
Transposons are a kind of movable genetic element and form a genetic unit which moves (transposes) from a portion of chromosomal DNA, plasmid DNA or viral DNA to another portion of the same or different DNA. They are widely distributed in bacteria, yeasts, maize, Drosophila, etc. The DNA site (target) to which they transpose is not fixed specifically, and it is presumed that they are able to transpose to any DNA site. Transposons are structurally characterized by an inverted repeat (IR) or a direct repeat at each end where recombination always occurs, indicating that this repeat has an important role in transposition. Transposons typically contain a gene responsible for the functions required for transposition or the expression of these functions.
Some transposons such as Tn3 and Tn1000 are known to function not only to bring about transposition of a gene but also to delete adjacent genes. Moreover, it was found that a mutation in the gene controlling transposition (tnpR) in a transposon increases the frequency of deletions as well as transposition reactions (Yoshinobu SUGINO and Hitoshi KAWASHIMA, Jpn. J. Genet. (1983) 58, pp. 79-93). A system for generating deletions in vivo has been developed (Sugino, Y. and Morita, M. 1994, Gene, 148, 169-170; and Wang, G. et al., Proc. Natl. Acad. Sci. USA (1993) 90, 7874-7878). This system is designed to insert a desired cloned DNA fragment into a vector having the terminal repeat of a transposon and transform said vector into E. coli which over expresses transposase to generate deletions from the terminal repeat of the transposon in E. coli. This system is commercially available as, for example, DELETION FACTORY.RTM. System from LIFE TECHNOLOGIES.
Although the conventional nested deletion methods are useful as described above, they involve problems due to in vivo reactions, e.g. they necessarily require a time for incubation and the desired gene may be denatured during reactions, etc. On the other hand, a transposon has been successfully transposed in vitro from a plasmid into .lambda. DNA (Ichikawa, H. and Ohtubo, E. 1990, J. Biol. Chem., 265,18829-18832). However, any method for generating nested deletions in vitro has not been known prior to the present invention, despite of a great need therefor.